Antisense oligonucleotides (ASOs) are synthetic nucleic acids that bind to a complementary target and suppress function of that target. Typically ASOs are used to reduce or alter expression of RNA targets, particularly messenger RNA (mRNA) or microRNA (miRNA) species. As a general principle, ASOs can suppress gene expression via two different mechanisms of action: 1) by steric blocking, wherein the ASO tightly binds the target nucleic acid and inactivates that species, preventing its participation in cellular activities, or 2) by triggering degradation, wherein the ASO binds the target and leads to activation of a cellular nuclease that degrades the targeted nucleic acid species. One class of “target degrading” ASOs is “RNase H active”, where formation of heteroduplex nucleic acids by hybridization of the target RNA with a DNA-containing RNase H active ASO forms a substrate for the enzyme RNase H. RNase H degrades the RNA portion of the heteroduplex molecule, thereby reducing expression of that species. Degradation of the target RNA releases the ASO, which is not degraded, and is then free to recycle and bind another RNA target of the same sequence. For an overview of antisense strategies, oligonucleotide design and chemical modifications, see Kurreck, 2003, Eur. J. Biochem., 270(8): 1628-44.
Unmodified DNA oligonucleotides have a half-life of minutes when incubated in human serum. Therefore, unmodified DNA oligonucleotides have limited utility as ASOs. The primary nuclease present in serum has a 3′-exonuclease activity (Eder et al., 1991, Antisense Res. Dev. 1(2): 141-51). Once an ASO gains access to the intracellular compartment, it is susceptible to endonuclease degradation. Historically, the first functional ASOs to gain widespread use comprised DNA modified with phosphorothioate groups (PS). PS modification of internucleotide linkages confers nuclease resistance, making the ASOs more stable both in serum and in cells. As an added benefit, the PS modification also increases binding of the ASO to serum proteins, such as albumin, which decreases the rate of renal excretion following intravenous injection, thereby improving pharmacokinetics and improving functional performance (Geary et al., 2001, Curr. Opin. Investig. Drugs, 2(4): 562-73). However, PS-modified ASOs are limited to a 1-3 day half-life in tissue, and the PS modifications reduce the binding affinity of the ASO for the target RNA, which can decrease potency (Stein et al., 1988, Nucleic Acids Res. 16(8): 3209-21).
The PS modification is unique in that it confers nuclease stability, yet still permits formation of a heteroduplex with RNA that is a substrate for RNase H. Most other modifications that confer nuclease resistance, such as methyl phosphonates or phosphoramidates, are modifications that do not form heteroduplexes that are RNase H substrates when hybridized to a target mRNA. Improved potency could be obtained using compounds that were both nuclease resistant and showed higher affinity to the target RNA, yet retain the ability to activate RNase H mediated degradation pathways.
Further design improvements were implemented to increase affinity for the target RNA while still maintaining nuclease resistance (see Walder et al., U.S. Pat. No. 6,197,944 for designs containing 3′-modifications with a region containing unmodified residues with phosphodiester linkages; see also European Patent No. 0618925 for “Gapmer” compounds having 2′-methoxyethylriboses (MOE's) providing 2′-modified “wings” at the 3′ and 5′ ends flanking a central 2′-deoxy gap region). This new strategy allows for chimeric molecules that have distinct functional domains. For example, a single ASO can contain a domain that confers both increased nuclease stability and increased binding affinity, but itself does not form an RNase H active substrate; a second domain in the same ASO can be RNase H activating. Having both functional domains in a single molecule improves performance and functional potency in antisense applications. One successful strategy is to build the ASO from different chemical groups, with a domain on each end intended to confer increased binding affinity and increased nuclease resistance, each flanking a central domain comprising different modifications. This facilitates RNase H activation. This so-called “end blocked” or “gapmer” design is the basis for the improved function “second generation” ASOs. Compounds of this design are typically significantly more potent as gene knockdown agents than the “first generation” PS-DNA ASOs.
Typically ASOs that function using steric blocking mechanisms of action show higher potency when made to maximize binding affinity. This can be accomplished through use of chemical modifications that increase binding affinity, such as many of the 2′-ribose modifications discussed herein, minor groove binders, or the internal non-base modifiers of the present invention. Alternatively, increased binding affinity can be achieved by using longer sequences. However, some targets are short, such as miRNAs, which are typically only 20-24 bases long. In this case, making ASOs longer to increase binding affinity is not possible. Furthermore, short synthetic oligonucleotides gain access into cells more efficiently than long oligonucleotides, making it desirable to employ short sequences with modifications that increase binding affinity (see, e.g., Straarup et al., 2010, Nucleic Acids Res. 38(20): 7100-11). The chemical modification and methods of the present invention enable synthesis of relatively short ASOs having increased binding affinity that show improved functional performance.
ASO modifications that improve both binding affinity and nuclease resistance typically are modified nucleosides that are costly to manufacture. Examples of modified nucleosides include locked nucleic acids (LNA), wherein a methyl bridge connects the 2′-oxygen and the 4′-carbon, locking the ribose in an A-form conformation; variations of LNA are also available, such as ethylene-bridged nucleic acids (ENA) that contain an additional methyl group, amino-LNA and thio-LNA. Additionally, other 2′-modifications, such as 2′-O-methoxyethyl (MOE) or 2′-fluoro (2′-F), can also be incorporated into ASOs. Some modifications decrease stability, and some can have negative effects such as toxicity (see Swayze et al., 2007, Nucleic Acids Res. 35(2): 687-700).
The present invention provides for non-nucleotide modifying groups that can be inserted between bases in an ASO to improve nuclease resistance and binding affinity, thereby increasing potency. The novel modifications of the present invention can be employed with previously described chemical modifications (such as PS internucleotide linkages, LNA bases, MOE bases, etc.) and with naturally occurring nucleic acid building blocks, such as DNA or 2′-O-Methyl RNA (2′OMe), which are inexpensive and non-toxic. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.